Surrogate therapeutic endpoint for anti-CTLA4-based immunotherapy of disease

ABSTRACT

The present invention provides a method of treatment using human sequence antibodies against human CTLA-4. In particular, methods of treating cancer are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application Ser.No. 60/475,067, filed on May 30, 2003, the contents of which are herebyincorporated in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to molecular immunology and thetreatment of human diseases. In particular, it relates to refinedtreatment methods using antibodies against human CTLA-4.

BACKGROUND OF THE INVENTION

The vertebrate immune system requires multiple signals to achieveoptimal immune activation (see, e.g., Janeway, Cold Spring Harbor Symp.Quant. Biol. 1989; 54:1-14; Paul William E., ed. Raven Press, N.Y.,Fundamental Immunology, 4th edition (1998), particularly chapters 12 and13, pages 411 to 478). Interactions between T lymphocytes (T cells) andantigen presenting cells (APC) are essential to the immune response.Levels of many cohesive molecules found on T cells and APC's increaseduring an immune response (Springer et al., A. Rev. Immunol. 1987;5:223-252; Shaw and Shimuzu, Current Opinion in Immnunology, 1988 Eds.Kindt and Long, 1:92-97; and Hemler, Immunology Today 1988; 9:109-113).Increased levels of these molecules may help explain why activated APC'sare more effective at stimulating antigen-specific T cell proliferationthan are resting APC's (Kaiuchi et al., J. Immunol. 1983; 131:109-114;Kreiger et al., J. Immunol. 1985; 135:2937-2945; McKenzie, J. Immunol.1988; 141:2907-2911; and Hawrylowicz and Unanue, J. Immunol. 1988;141:4083-4088).

T cell immune response is a complex process that involves cell-cellinteractions (Springer et al., A. Rev. Immunol. 1987; 5:223-252),particularly between T and accessory cells such as APC's, and productionof soluble immune mediators (cytokines or lymphokines) (Dinarello, NewEngl. J. Med 1987; 317:940-945; Sallusto, J. Exp. Med. 1997;179:1109-1118). This response is regulated by several T-cell surfacereceptors, including the T-cell receptor complex (Weiss, Ann. Rev.Immunol. 1986; 4:593-619) and other “accessory” surface molecules(Allison, Curr. Opin. Immunol. 1994; 6:414-419; Springer, 1987, supra).Many of these accessory molecules are naturally occurring cell surfacedifferentiation (CD) antigens defined by the reactivity of monoclonalantibodies on the surface of cells (McMichael, Ed., Leukocyte TypingIII, Oxford Univ. Press, Oxford, N.Y., 1987).

CD28 antigen, a homodimeric glycoprotein of the immunoglobulinsuperfamily (Aruffo and Seed, Proc. Natl. Acad. Sci. 1987;84:8573-8577), is an accessory molecule found on most mature human Tcells (Damle et al., J. Immunol. 1983; 131:2296-2300). Current evidencesuggests that this molecule functions in an alternative T cellactivation pathway distinct from that initiated by the T-cell receptorcomplex (June et al., Mol. Cell. Biol. 1987; 7:4472-4481). Monoclonalantibodies (MAbs) reactive with CD28 antigen can augment T cellresponses initiated by various polyclonal stimuli (reviewed by June etal., supra). These stimulatory effects may result from MAb-inducedcytokine production (Thompson et al., Proc. Natl. Acad. Sci 1989;86:1333-1337; and Lindsten et al., Science 1989; 244:339-343) as aconsequence of increased mRNA stabilization (Lindsten et al., 1989,supra).

CTLA-4 is accepted as opposing CD28 activity and dampening T cellactivation (Krummel, J. Exp. Med. 1995; 182:459-465; Krummel et al.,Int'l Immunol. 1996; 8:519-523; Chambers et al., Immunity. 1997;7:885-895). CTLA-4 deficient mice suffer from massivelymphoproliferation (Chambers et al., supra). It has been reported thatCTLA-4 blockade augments T cell responses in vitro (Walunas et al.,Immunity. 1994; 1:405-413) and in vivo (Kearney, J. Immunol. 1995;155:1032-1036), exacerbates antitumor immunity (Leach, Science 1996;271:1734-1736), and enhances an induced autoimmune disease (Luhder, JExp. Med. 1998; 187:427-432). It has also been reported that CTLA-4 hasan alternative or additional impact on the initial character of the Tcell immune response (Chambers, Curr. Opin. Immunol. 1997; 9:396-404;Bluestone, J. Immunol. 1997; 158:1989-1993; Thompson, Immunity 1997;7:445-450). This is consistent with the observation that some autoimmunepatients have autoantibodies to CTLA-4. It is possible that CTLA-4blocking autoantibodies play a pathogenic role in these patients(Matsui, J. Immunol. 1999; 162:4328-4335).

Non-human CTLA-4 antibodies have been used in the various studiesdiscussed above. Furthermore, human antibodies against human CTLA-4 havebeen described as immunostimulation modulators in a number of diseaseconditions, such as treating or preventing viral and bacterial infectionand for treating cancer (e.g., PCT Publication WO 01/14424 and PCTPublication WO 00/37504). U.S. Pat. No. 5,855,887 discloses a method ofincreasing the response of a mammalian T cell to antigenic stimulationby combining a T cell with a CTLA-4 blocking agent. U.S. Pat. No.5,811,097 discloses a method of decreasing the growth of non-T celltumors by administering a CTLA-4 blocking agent. U.S. patent applicationSer. Nos. 09/644,668 and 09/948,939 disclose human CTLA-4 antibodies.Each of these patents and applications is hereby incorporated byreference.

The citation or discussion of any reference in this section or elsewherein the specification is made only to clarify the description of thepresent invention and is not an admission that any such reference is“prior art” to any invention described herein.

SUMMARY OF THE INVENTION

The present invention provides a novel method of treating cancer in apatient by administering anti-CTLA-4 antibody to the patient in a dosagesufficient to induce a breakthrough event and detecting the breakthroughevent in the patient. In one embodiment the breakthrough event is anautoimmune response to an antigen that exists on non-cancer cells. Inanother embodiment the breakthrough event is grade 3 or 4. According tothe methods of the invention, the breakthrough event is induced byadministering escalating dosage amounts of anti-CTLA-4 antibody. In afurther embodiment, the breakthrough event is induced by administeringanti-CTLA-4 antibody at a reduced dosage interval. In anotherembodiment, the breakthrough event is a manifestation of non-tumorrelated autoimmunity. The present invention provides methods foradministering a therapeutically effective dosage regimen of anti-CTLA-4antibody for the treatment of cancer. The invention also providesmethods for determining such dosage regimens.

All publications, figures, GenBank Accession references (sequences),ATCC Deposits, patents and patent applications cited herein are herebyexpressly incorporated by reference for all purposes to the same extentas if each was so individually denoted.

DETAILED DESCRIPTION

The present invention provides more effective, predictable CTLA-4antibody-based methods for cancer treatment. The methods of theinvention represent a significant development in the treatment ofcancers with anti-CTLA-4 antibody because they avoid under-dosingpatients with the antibody. A statistically significant correlationbetween the appearance of an adverse event and response to treatment hasbeen unexpectedly discovered in cancer patients treated with anti-CTLA-4antibody. Whereas avoidance of serious “adverse events” has been a goalin the treatment of seriously ill cancer patients, the methods of theinvention have the goal of inducing and detecting these events (hereintermed “breakthrough events” (BE)). The induction of a significant, butreversible, breakthrough event is medically sound in these patientsbecause treatment with a sufficient dose of anti-CTLA-4 antibody mayimprove the patient's cancer or prolong life.

BE's are acute, dose-related, easily monitored and, to some extent,predictable. BE's can be reversed with drug withdrawal and/or supportivecare with or without specific treatment, usually corticosteroid therapy.Administering a dosage of anti-CTLA-4 antibody to induce a BE anddetecting the BE is an effective method to treat cancer because of thesignificant correlation between response to anti-CTLA-4 antibodytreatment and the development of a BE.

In a particular embodiment, the anti-CTLA-4 antibody of the presentinvention is human monoclonal antibody 10D1 as disclosed in WO 01/14424.

The present invention is based, in part, on observations made duringclinical testing of a human sequence anti-CTLA-4 antibody inimmunotherapy of cancers, as described below. The tests demonstrate theeffectiveness of anti-CTLA antibody in the treatment of patients withcancer when a dosage of anti-CTLA-4 antibody sufficient to induce a BEis delivered. According to the invention, anti-CTLA-4 antibody isadministered in a dosage sufficient to induce a BE and the BE isdetected in the patient.

Various studies led to recognition of a correlation between BE's andanti-tumor efficacy. For example, in cohort 1 of a study, fourteenpatients with Stage IV melanoma received anti-CTLA-4 antibody 10D1(MDX-010) at 3 mg/kg every three weeks for eight weeks in conjunctionwith vaccination with two gp100 peptides. All patients had prior surgeryfor their primary tumor. Six patients had prior chemotherapy. Elevenpatients had prior immunotherapy. Clinical response was measured bycomputed axial tomography (CT) and magnetic resonance (MR) imaging.Patient 11, who had prior chemotherapy, had complete resolution of lung,brain and subcutaneous tumors after 5 treatment cycles. Patient 13, whohad prior chemotherapy and immunotherapy, had complete resolution ofadrenal and lung tumors. Patient 1, who had prior chemotherapy andimmunotherapy, was a partial responder. Each of the three respondersexperienced a grade 3 BE. Patient 1, a partial responder had grade 3enterocolitis and dermatitis. Patient 1 was treated for autoimmuneenterocolitis with IV methylprednisolone, which resulted with markedimprovement within 24 hours. Patient 11, a complete responder, had grade3 hypophysitis and panhypopituitarism. Patient 11 received replacementdoses of thyroxine, testosterone and hydrocortisone. Patient 13, acomplete responder, had grade 3 dermatitis that resolved upon treatmentwith hydoxyzine. This study unexpectedly showed that all threeresponders experienced grade 3 adverse events.

In cohort 2 of the study, twenty-four patients with unresectedmetastatic melanoma were administered anti-CTLA-4 antibody 10D1 with aninitial loading dose of 3 mg/kg and subsequent doses of 1 mg/kg every 3weeks in combination with gp100 peptide vaccines. To date, 3 of 24patients (13%) have had objective tumor responses. One of the threeresponders had a grade 3 adverse event (diarrhea).

A statistically significant correlation was discovered in cohorts 1 and2 between patients that respond and patients that develop seriousadverse events, using both the Chi-square test (p=0.0146) and theFisher's exact test (p=0.0116). See Fisher and Van Belle, 1993,Biostatistics: A methodology for the Health Sciences, J. Wiley and Sons,New York.

In another study, seventeen patients with Stage III or IV malignantmelanoma were administered a single dose of anti-CTLA-4 antibody 10D1.Two patients had a partial response. There were no serious (grade 3 or4) adverse events.

Thirteen patients with malignant melanoma were administered anti-CTLA-4antibody 10D1 (3 mg/kg×2 doses 8 weeks apart) in combination with theapproved regimen for MELACINE® (including cyclophosphamide). Noobjective responses and no serious adverse events were observed.Possible reasons for lack of efficacy include: (1) inhibitory effects ofcyclophosphamide, (2) the long dosing interval, and (3) the weak potencyof MELACINE® as a vaccine.

In combining the results of the above studies, a statisticallysignificant correlation was discovered between patients who respond andpatients who develop serious adverse events, using both the Chi-squaretest (p=0.0028) and the Fisher's exact test (p=0.0049).

In a study of nineteen patients with completely resected stage III or IVmelanoma, the patients were divided into three cohorts and treated withdifferent doses of anti-CTLA-4 antibody 10D1 (0.1, 1.0 and 3.0 mg/kgmonthly for 6 months, then every 3 months×2) in combination with gp100,tyrosinase, and MART-1 vaccines. This study showed dose-dependentinduction of organ specific autoimmune-like adverse events,predominately involving skin and gut. The autoimmune-like adverse eventswere manageable and reversible.

In a study of treatment of chemotherapy-naïve patients with metastaticmelanoma, patients were treated with anti-CTLA-4 antibody 10D1 alone orin combination with cytotoxic chemotherapy (dacarbazine). Twenty-fourpatients enrolled. Four of twelve patients receiving monotherapy andthree of twelve patients receiving combination treatment experienceddisease progression. One SAE of grade 3 rash and pruritis was observed.

Administration of anti-CTLA-4 antibody has been associated with seriousadverse events that are suggestive of autoimmune responses. SAE's wereinfrequent following a single dose of anti-CTLA-4 antibody given aloneat 3 mg/kg. Adverse events occur more often when anti-CTLA-4 antibody isgiven in multiple doses and in combination with melanoma peptidevaccines. In the study involving administration of anti-CTLA-4 antibodyin conjunction with peptide vaccines, reduction in the dose ofanti-CTLA-4 antibody in cohort 2 reduced the rate of SAE's. Nodiscernable correlation between plasma concentration of anti-CTLA-4antibody in an individual patient and the development of SAE's has beenfound. Statistical analysis of the clinical trial data established anunexpected but highly significant correlation between a BE andtherapeutic efficacy. This experimental observation underpins thediscovery that dosing anti-CTLA-4 antibody up to the point of inducing aBE (either by the dosage amount, frequency, or both) indicatesachievement of a therapeutically maximally effective dose.

Except when noted, the terms “patient” or “subject” are usedinterchangeably and refer to mammals such as human patients andnon-human primates, as well as experimental animals such as rabbits,rats, and mice, and other animals. Animals include all vertebrates,e.g., mammals and non-mammals, such as sheep, dogs, cows, chickens,amphibians, and reptiles.

An “adverse event” (AE) as used herein is any unfavorable and generallyunintended, even undesirable, sign (including an abnormal laboratoryfinding), symptom, or disease associated with the use of a medicaltreatment or procedure. Most AE's are temporary and reverse uponwithdrawal or reduction in dose of the medical treatment, or withtreatment of the AE.

The National Cancer Institute defines “adverse event” as any unfavorableand unintended sign (including an abnormal laboratory finding), symptom,or disease temporally associated with the use of a medical treatment orprocedure that may or may not be considered related to the medicaltreatment or procedure (Cancer Therapy Evaluation Program, CommonTerminology Criteria for Adverse Events, Version 3.0, DCTD, NCI, NIH,DHHS, Mar. 31, 2003 (http://ctep.cancer.gov), published Apr. 16, 2003(site visited May 27, 2003)). An “adverse event” is an unintendedconsequence of treatment. It has been surprisingly discovered thatinduction of an adverse event is a marker that indicates a sufficientdosage of anti-CTLA-4 antibody has been administered to a patient forthe treatment of cancer. In the context of this invention, the “adverse”events are not unintended, but rather are purposely sought because theyserve as a surrogate therapeutic endpoint for anti-CTLA-4 basedimmunotherapy of cancer. Because the signs, symptoms, abnormallaboratory findings and diseases temporally associated with anti-CTLA-4treatment are an intended consequence of treatment by the methods of theinvention, such events are herein referred to as “breakthrough events(BE's)”.

A “breakthrough event (BE)” as used herein is an intended sign(including an abnormal laboratory finding), symptom, or diseasetemporally associated with administration of anti-CTLA-4 antibody thatis apart from the therapeutic effect at the tumor site. For example, anautoimmune response that causes dermatitis at a location separate inspace from the melanoma under treatment is a BE. A BE is generally anautoimmune event but, for the purposes of the use of the invention inclinical practice, pathological confirmation of an autoimmune etiologyis not required; for example, colitis diagnosed clinically of anyetiology can be a breakthrough event if the definition is otherwisesatisfied. A BE can be graded according to the NCI grading system foradverse events.

An “autoimmune breakthrough event (ABE)” is a breakthrough event that isan autoimmune event. When practicing the present invention preferredbreakthrough events are autoimmune breakthrough events. Accordingly, theterm “autoimmune breakthrough event” is frequently used to describe thetherapeutic methods of this invention. It is understood, however, thatthe breakthrough events that are useful to such methods need notnecessarily be autoimmune events. An autoimmune breakthrough event is apreferred breakthrough event.

A “serious adverse event” (SAE) is a grade 3 or 4 adverse event asdefined by the National Cancer Institute (NCI). A grade 3 AE isgenerally defined as “severe” and a grade 4 AE is generally defined as“life-threatening or disabling”. The NCI also specifically defines grade3 and 4 adverse events. For example, grade 3 colitis consists ofabdominal pain, fever, change in bowel habits with ileus, or peritonealsigns (Cancer Therapy Evaluation Program, Common Terminology Criteriafor Adverse Events, Version 3.0, DCTD, NCI, NIH, DHHS, Mar. 31, 2003,available at the National Cancer Institute's Cancer Therapy EvaluationProgram (CTEP) web site, publication date Apr. 16, 2003. Thispublication is hereby incorporated by reference.

A “manifestation of non-tumor related autoimmunity” is any clinicalevent that results from, or appears to result from, immune targeting ofantigens on non-cancer cells. Such a BE is particularly indicative of animmunologically mediated therapeutic effect on the cancer cells, sincethe BE relates to a heightened activation of overall immunity includingtumor immunity.

The term “treating” includes the administration of the compounds oragents of the present invention to prevent or delay the onset of thesymptoms, complications, or biochemical indicia of a disease,alleviating the symptoms or arresting or inhibiting further developmentof the disease, condition, or disorder (e.g., autoimmune disease).Treatment may be prophylactic (to prevent or delay the onset of thedisease, or to prevent the manifestation of clinical or subclinicalsymptoms thereof) or therapeutic suppression or alleviation of symptomsafter the manifestation of the disease.

The term “advanced cancer” means cancer that is no longer localized tothe primary tumor site, or a cancer that is Stage III or IV according tothe American Joint Committee on Cancer (AJCC).

The term “therapeutically effective dose” means a dose of anti-CTLA-4antibody sufficient to induce a cancer to shrink, to slow theprogression of a cancer or to stop the progression of a cancer.Alternatively, a “therapeutically effective dose” means a dose ofanti-CTLA-4 antibody sufficient to induce a partial or a completeresponse in a patient with cancer.

The term “lymphocyte” as used herein has the normal meaning in the art,and refers to any of the mononuclear, nonphagocytic leukocytes, found inthe blood, lymph, and lymphoid tissues, i.e., B and T lymphocytes.

The terms “cytotoxic T lymphocyte-associated antigen-4,” “CTLA-4,”“CTLA4,” “CTLA-4 antigen” and “CD152” (see, e.g., Murata (1999) Am. J.Pathol. 155:453-460) are used interchangeably, and include variants,isoforms, species homologs of human CTLA-4, and analogs having at leastone common epitope with CTLA-4 (see, e.g., Balzano (1992) Int. J. CancerSuppl. 7:28-32). CTLA-4's complete sequence is found in GenBankAccession No. L15006.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

An intact “antibody” comprises at least two heavy (H) chains and twolight (L) chains inter-connected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as HCVRor VH) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. Each light chainis comprised of a light chain variable region (abbreviated herein asLCVR or VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (Clq) of the classicalcomplement system. The term antibody includes antigen-binding portionsof an intact antibody that retain capacity to bind CTLA-4. Examples ofbinding include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (scFv); See, e.g., Bird et al., Science 1998; 242:423-426; and Hustonet al., Proc. Natl. Acad. Sci. USA 1988; 85:5879-5883). Such singlechain antibodies are included by reference to the term “antibody”Fragments can be prepared by recombinant techniques or enzymatic orchemical cleavage of intact antibodies.

CTLA-4 antibodies can bind to an epitope on human CTLA-4 so as toinhibit CTLA-4 from interacting with a human B7 counterreceptor. Becauseinteraction of human CTLA-4 with human B7 transduces a signal leading toinactivation of T-cells bearing the human CTLA-4 receptor, antagonism ofthe interaction effectively induces, augments or prolongs the activationof T cells bearing the human CTLA-4 receptor, thereby prolonging oraugmenting an immune response. Anti-CTLA-4 antibodies are described inU.S. Pat. Nos. 5,811,097; 5,855,887; 6,051,227; in PCT Publication Nos.WO 01/14424 and WO 00/37504; and in U.S. Publication No. 2002/0039581A1. Each of these references is specifically incorporated herein byreference for purposes of description of anti-CTLA-4 antibodies. Apreferred clinical anti-CTLA-4 antibody is human monoclonal antibody10D1 (MDX010) as disclosed in WO 01/14424.

The phrase “immune cell response” refers to the response of immunesystem cells to external or internal stimuli (e.g., antigen, cytokines,chemokines, and other cells) producing biochemical changes in the immunecells that result in immune cell migration, killing of target cells,phagocytosis, production of antibodies, other soluble effectors of theimmune response, and the like.

The terms “T lymphocyte response” and “T lymphocyte activity” are usedhere interchangeably to refer to the component of immune responsedependent on T lymphocytes (i.e., the proliferation and/ordifferentiation of T lymphocytes into helper, cytotoxic killer, orsuppressor T lymphocytes, the provision of signals by helper Tlymphocytes to B lymphocytes that cause or prevent antibody production,the killing of specific target cells by cytotoxic T lymphocytes, and therelease of soluble factors such as cytokines that modulate the functionof other immune cells).

The term “immune response” refers to the concerted action oflymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

As used herein, the phrase “cell surface receptor” includes moleculesand complexes of molecules capable of receiving a signal and thetransmission of such a signal across the plasma membrane of a cell. Anexample of a “cell surface receptor” of the present invention is the Tcell receptor (TCR) or the B7 ligands of CTLA-4.

The term “nonspecific T cell activation” refers to the stimulation of Tcells independent of their antigenic specificity.

“Target cell” shall mean any undesirable cell in a subject (e.g., ahuman or animal) that can be targeted by a composition (e.g., a humansequence antibody or a human monoclonal antibody of the invention, abispecific or a multispecific molecule of the invention). The targetcell can be a cell expressing or overexpressing human CTLA-4. Cellsexpressing human CTLA-4 can include tumor cells, e.g. lymphomas.

Also included in the invention are modified antibodies. The term“modified antibody” includes antibodies, such as monoclonal antibodies,chimeric antibodies, and humanized antibodies which have been modifiedby, e.g., deleting, adding, or substituting portions of the antibody.For example, an antibody can be modified by deleting the constant regionand replacing it with a constant region meant to increase half-life,e.g., serum half-life, stability or affinity of the antibody.

The antibody conjugates of the invention can be used to modify a givenbiological response or create a biological response (e.g., to recruiteffector cells). The drug moiety is not to be construed as limited toclassical chemical therapeutic agents. For example, the drug moiety maybe a protein or polypeptide possessing a desired biological activity.Such proteins may include, for example, an enzymatically active toxin,or active fragment thereof, such as abrin, ricin A, pseudomonasexotoxin, or diphtheria toxin; a protein such as tumor necrosis factoror interferon-alpha; or, biological response modifiers such as, forexample, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Inducement of a Breakthrough Event

The objective of the present method is to achieve the BE in the shortestamount of time while avoiding overdosage. This is accomplished bytitrating the dosage of medication to induce the BE. Methods oftitrating medication dosage to achieve a desired effect are well-knownin the medical art as, for example, in the treatment of hypertension inwhich the dosage of anti-hypertensive medication is titrated to achievethe desired effect on blood pressure. Such methods include, for example,repeating the same dosage amount of a medication at a fixed dosageinterval, but more preferably increasing the dosage amount, decreasingthe dosage interval, or a combination of altering dosage amount anddosage interval.

An initial dose of anti-CTLA-4 antibody typically comprises 3 to 10mg/kg antibody administered every 3 to 8 weeks. Following an initialdose of anti-CTLA-4 antibody, a patient is monitored by a clinician fora sufficient period of time, which is typically over the course of thedosage interval or 1 to 4 weeks, to detect a BE (see below for methodsof detection). Since a BE may require expansion of autoreactive T-cells,it is expected that this event could take 1-4 weeks to manifest afterany therapeutically effective dose. The absence of a BE during themonitoring period is an indication to the clinician that furtheradministration of anti-CTLA-4 antibody is required. Following a dose ofanti-CTLA-4 antibody, the patient is monitored for a BE, and additionaldosages are administered until, at least, a BE is induced and detected.It shall be appreciated by those of skill in the art that factors,including the patient's immune state, which may be affected by priorimmune therapies, disease state, age, etc., can impact the dosagerequired to elicit a BE. A skilled clinician will be able to take suchfactors into account when determining the initial dose, as well as anysubsequent doses, to induce a BE. These factors should be considered indetermining the initial dose, as well as subsequent dosages, to induce aBE.

Since a method of the invention is to induce a BE in the shortest periodof time without causing an overdose in the patient, it is advantageousto shorten the period for monitoring the patient for a BE to 1 to 4weeks, more preferably 2 to 3 weeks. If an additional dose is requiredto achieve a BE, the dosage can be increased by, e.g., 10 to 100% of theprior dosage. For example, if the patient initially receives 3 mg/kg andis scheduled to receive a second dose 3 weeks later, but does notachieve a BE after the 3 week period, then the patient's dosage can beincreased to 6 mg/kg (i.e., 100% increase). In another example, if thepatient initially receives 10 mg/kg and is scheduled to receive a seconddose 8 weeks later, but does not achieve a BE after monitoring for 4weeks, then the patient's dosage interval can be reduced from 8 weeks to4 weeks, while maintaining the 10 mg/kg dosage. One skilled in the artshall appreciate that various dosage amount and interval changes can bemade in practicing a method of the invention to induce a BE.

Detecting a Breakthrough Event

A BE can manifest as a symptom, sign, or laboratory abnormality.Accordingly, detection of such an event requires a patient history (forsubjective complaints), a physical examination and/or imaging studies(for objective signs), and laboratory studies (for laboratoryabnormalities). Depending on the initial findings, a clinician may electto order additional studies such as, for example, an endoscopy or abiopsy.

A clinician is particularly vigilant regarding detection of more commonBE's such as, for example, those involving the skin (dermatitis, rash,pruritis), gastrointestinal tract (abdominal pain, tenderness,diarrhea), endocrine system (suppression of hormone levels), and liver(hepatitis, elevation of liver function tests). The clinician, however,must be prepared to detect any BE. The Cancer Therapy EvaluationProgram, Common Terminology Criteria for Adverse Events lists BE's(adverse events) and provides a grading system for these events. Thispublication can also serve as a guide to aid the clinician in themonitoring patients and detecting BE's.

Patient monitoring includes periodic comprehensive histories forsubjective complaints and comprehensive physical examination. Each organsystem susceptible to a BE is examined. For example, the history relatedto the dermatologic system includes questions regarding itching,scaling, pain, and changes in skin color. The physical examination ofthe skin includes, for example, close inspection of all the visibledermis. Further studies, based on the judgment of the clinician, caninclude, for example, skin biopsy.

BE's in organ systems that are not readily amenable to physicalexamination, and that are asymptomatic, require added reliance onlaboratory studies and imaging studies. For example, hepatitis may bedetected at earlier stage using liver function tests and CT scan than byphysical examination.

The detection of BE's is dependent on clinical monitoring of patientsfollowing administration of anti-CTLA-4 antibody. A broad array ofdiagnostic methods may be used by the clinician to detect a BE.

Cancer Treatment

The CTLA-4 antibodies of the invention and surrogate therapeuticendpoint can be used in the treatment of malignancies, where the patienthas previously received a cancer vaccine or demonstrates some level ofnatural protective immunity to the tumor. The antibodies can be used asa single agent or in combination with one or more other therapeuticagents or in conjunction with an immunotherapeutic vaccine for thetumor, such as chemotherapy, radiation therapy, cytokines, chemokinesand other biologic signaling molecules, tumor specific vaccines,autologous and allogeneic stem cell rescue (e.g., to augment graftversus tumor effects), other therapeutic antibodies, molecular targetedtherapies, anti-angiogenic therapy, infectious agents with therapeuticintent (such as tumor localizing bacteria), and gene therapy. Theantibodies can be administered as a single dose or as multiple doses.The antibodies can be used in adjuvant or neoadjuvant therapy, eitheralone or in conjunction with the aforementioned therapies.

A therapeutic agent, which is intended to treat the BE, e.g., steroidscan also be used in a method of the invention. Thus, a therapeutic agentthat treats the BE is administered to the patient following diagnosis ofthe BE.

The present invention is directed to the treatment of tumors,particularly immunologically sensitive tumors, which are cancers thatrespond to immunotherapy or cancers that manifest in patients who areimmunocompromised. In one embodiment the tumor is a solid tumor.Examples of tumors that can be treated according to the inventioninclude sarcomas and carcinomas such as, but not limited to:fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, lymphoma,melanoma, Kaposi's sarcoma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colo-rectal carcinoma, gastric carcinoma, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, testicular tumor, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

In another embodiment, dysproliferative changes (such as metaplasias anddysplasias) are treated or prevented in epithelial tissues such as thosein the cervix, esophagus, and lung. Thus, the present invention providesfor treatment of conditions known or suspected of preceding progressionto neoplasia or cancer, in particular, where non-neoplastic cell growthconsisting of hyperplasia, metaplasia, or most particularly, dysplasiahas occurred (for review of such abnormal growth conditions, see Robbinsand Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co.,Philadelphia, pp. 68-79). Hyperplasia is a form of controlled cellproliferation involving an increase in cell number in a tissue or organ,without significant alteration in structure or function. As but oneexample, endometrial hyperplasia often precedes endometrial cancer.Metaplasia is a form of controlled cell growth in which one type ofadult or fully differentiated cell substitutes for another type of adultcell. Metaplasia can occur in epithelial or connective tissue cells.Atypical metaplasia involves a somewhat disorderly metaplasticepithelium. Dysplasia is frequently a forerunner of cancer, and is foundmainly in the epithelia; it is the most disorderly form ofnon-neoplastic cell growth, involving a loss in individual celluniformity and in the architectural orientation of cells. Dysplasticcells often have abnormally large, deeply stained nuclei, and exhibitpleomorphism. Dysplasia characteristically occurs where there existschronic irritation or inflammation, and is often found in the cervix,respiratory passages, oral cavity, and gall bladder. For a review ofsuch disorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B.Lippincott Co., Philadelphia.

The present invention is also directed to treatment of non-malignanttumors and other disorders involving inappropriate cell or tissue growthaugmented by angiogenesis by administering a therapeutically effectiveamount of a vector of the invention to the tissue undergoinginappropriate growth. For example, it is contemplated that the inventionis useful for the treatment of arteriovenous (AV) malformations,particularly in intracranial sites. The invention may also be used totreat psoriasis, a dermatologic condition that is characterized byinflammation and vascular proliferation; and benign prostatichypertrophy, a condition associated with inflammation and possiblyvascular proliferation. Treatment of other hyperproliferative disordersis also contemplate

Treatment with an anti-CTLA-4 antibody can be used to activate apre-existing memory response in patients treated with a cancer vaccine.Thus, vaccine-treated patients can be selected for further treatmentwith an anti-CTLA-4 antibody to thereby further induce or enhance animmune response.

In one embodiment, the patient has been previously treated with ananti-cancer vaccine. The cancer antigen can be, for example, a melanomaantigen or a prostate cancer antigen. In one embodiment, the patient isa human. In a preferred embodiment, the anti-CTLA-4 antibody is a humananti-CTLA-4 antibody. A preferred human anti-CTLA-4 antibody of theinvention is 10D 1, but the methods of the present invention can be usedwith any human CTLA-4 antibody. In other embodiments, the anti-CTLA-4antibody is a recombinant antibody such as a chimeric or humanized(e.g., CDR-grafted) anti-CTLA-4 antibody.

Blockade of CTLA-4 by antibodies can enhance the memory or secondaryimmune response to cancerous cells in the patient. Antibodies to CTLA-4can be combined with an immunogenic agent, such as cancerous cells,purified tumor antigens (including recombinant proteins, peptides, andcarbohydrate molecules), cells, and cells transfected with genesencoding immune stimulating cytokines and cell surface antigens such asB7 (see, e.g., Hurwitz, A. et al. (1998) Proc. Natl. Acad. Sci U.S.A.1998; 95:10067-10071), or used alone, to stimulate immunity.

CTLA-4 blockade is effective when following a vaccination protocol. Manyexperimental strategies for vaccination against tumors have been devised(see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCOEducational Book Spring: 60-62; Logothetis, C., 2000, ASCO EducationalBook Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring:414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see alsoRestifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 inDeVita, V. et al. (eds.), 1997, Cancer: Principles and Practice ofOncology, Fifth Edition). In one of these strategies, a vaccine isprepared using autologous or allogeneic tumor cells. These cellularvaccines have been shown to be most effective when the tumor cells aretransduced to express GM-CSF. GM-CSF has been shown to be a potentactivator of antigen presentation for tumor vaccination (Dranoff et al.Proc. Natl. Acad. Sci U.S.A. 1993; 90: 3539-43).

Anti-CTLA-4 blockade to boost GMCSF-modified tumor cell vaccinesimproves efficacy of vaccines in a number of experimental tumor modelssuch as mammary carcinoma (Hurwitz et al., 1998, supra), primaryprostate cancer (Hurwitz et al., Cancer Research 2000; 60:2444-8) andmelanoma (van Elsas et al. J. Exp. Med. 1999, 190:355-66). In theseinstances, non-immunogenic tumors, such as the B16 melanoma, have beenrendered susceptible to destruction by the immune system. The tumor cellvaccine may also be modified to express other immune activators such asIL2, and costimulatory molecules, among others.

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called “tumor specificantigens” (Rosenberg, Immunity 1999; 10:281-7). In many cases, thesetumor specific antigens are differentiation antigens expressed in thetumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, Trp-2. More importantly, manyof these antigens can be shown to be the targets of tumor specific Tcells found in the host. CTLA-4 blockade may be used as a boosting agentin conjunction with vaccines based on recombinant versions of proteinsand/or peptides found to be expressed in a tumor in order to potentiatea secondary or memory immune response to these proteins. These proteinsare normally viewed by the immune system as self antigens and aretherefore tolerant to them. The tumor antigen may also include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim et al., Science 1994;266:2011-2013). These somatic tissues may be protected from immuneattack by various means. Tumor antigen may also be “neo-antigens”expressed in cancer cells because of somatic mutations that alterprotein sequence or create fusion proteins between two unrelatedsequences (i.e. bcr-abl in the Philadelphia chromosome), or idiotypefrom B cell tumors. Other tumor vaccines may include the proteins fromviruses implicated in human cancers such a Human Papilloma Viruses(HPV), Hepatitis Viruses (HBV and HCV) and Kaposi's Herpes Sarcoma Virus(KHSV). Another form of tumor specific antigen which may be used inconjunction with CTLA-4 blockade is purified heat shock proteins (HSP)isolated from the tumor tissue itself. These heat shock proteins containfragments of proteins from the tumor cells and these HSPs are highlyefficient at delivery to antigen presenting cells for eliciting tumorimmunity (Suot and Srivastava, Science 1995; 269:1585-1588; Tamura etal., Science 1997, 278:117-120.

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al., Nature Medicine 1998; 4:328-332). DCs mayalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al., Nature Medicine 2000; 6:332-336). As amethod of vaccination, DC immunization may be effectively boosted withCTLA-4 blockade to activate more potent anti-tumor responses.

Another type of melanoma vaccine that can be combined with CTLA-4blockade is a vaccine prepared from a melanoma cell line lysate, inconjunction with an immunological adjuvant, such as the MELACINE®vaccine, a mixture of lysates from two human melanoma cell lines plusDETOX™ immunological adjuvant. Vaccine treatment can be boosted withanti-CTLA4, with or without additional chemotherapeutic treatment.

Infectious Diseases

Other methods of the invention are used to treat patients that have beenexposed to particular toxins or pathogens. Similar to its application totumors as discussed above, antibody mediated CTLA-4 blockade andsurrogate therapeutic endpoint can be used alone, or as an adjuvant, incombination with vaccines, to stimulate the secondary or memory immuneresponse to pathogens, toxins, and self-antigens. CTLA-4 blockade hasbeen shown to be effective in the acute phase of infections ofNippostrongylus brasiliensis (McCoy, K. et al. (1997) 186(2); 183-187)and Leishmania donovani (Murphy, M. et al. (1998) J. Immunol.161:4153-4160). Examples of pathogens for which this therapeuticapproach may be particularly useful include pathogens for which there iscurrently no effective vaccine, or pathogens for which conventionalvaccines are less than completely effective. These include, but are notlimited to HIV, Hepatitis (A, B, & C), Influenza, Herpes, Giardia,Malaria, Leishmania, Staphylococcus aureus, and Pseudomonas aeruginosa.CTLA-4 blockade is particularly useful in boosting immunity againstestablished infections by agents such as HIV that present alteredantigens over the course of the infections. These novel epitopes arerecognized as foreign at the time of anti-human CTLA-4 administration,thus provoking a strong T cell response that is not dampened by negativesignals through CTLA-4.

Some examples of pathogenic viruses causing infections treatable bymethods of the invention include hepatitis (A, B, or C), herpes virus(e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods of the invention include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand conococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lyme disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods of the invention include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (Mucor, Absidia, Rhizophus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods of the invention include Entamoeba histolytica, Balantidiumcoli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondi, Nippostrongylus brasiliensis.

Promoting Beneficial Autoimmune Reactions

The ability of anti-CTLA-4 antibodies and surrogate therapeutic endpointto provoke and amplify autoimmune responses has been documented in anumber of experimental systems (EAE—Experimental AutoimmuneEncephalomyelitis, a murine model for MS (Perrin et al., J Immunol 1996;157:1333-1336); diabetes (Luhder et al., 1998, supra). Indeed, inductionof anti-tumor responses using tumor cell and peptide vaccines revealsthat many anti-tumor responses involve anti-self reactivities(depigmentation observed in anti-CTLA-4+GM-CSF modified B16 melanoma invan Elsas et al. supra; depigmentation in Trp-2 vaccinated mice(Overwijk et al., Proc. Natl. Acad. Sci. U.S.A. 1999 96:2982-2987);autoimmune prostatitis evoked by TRAMP tumor cell vaccines (Hurwitz2000, supra), melanoma peptide antigen vaccination and vitiligo observedin human clinical trials (Rosenberg and White, J Immunother EmphasisTumor Immunol 1996; 19: 81-4).

Therefore, it is possible to consider using anti-CTLA-4 boosting inconjunction with various self-proteins in order to devise vaccinationprotocols to efficiently generate immune responses against theseself-proteins for disease treatment. For example, Alzheimer's diseaseinvolves inappropriate accumulation of Aβ peptide in amyloid deposits inthe brain; antibody responses against amyloid are able to clear theseamyloid deposits (Schenk et al., Nature 1999; 400:173-177).

Other self-proteins may also be used as targets such as IgE for thetreatment of allergy and asthma, and TNF for rheumatoid arthritis.Finally, antibody responses to various hormones may be induced by theuse of anti-CTLA-4 antibody. Neutralizing antibody responses toreproductive hormones may be used for contraception. Neutralizingantibody response to hormones and other soluble factors that arerequired for the growth of particular tumors may also be considered aspossible vaccination targets.

Analogous methods as described above for the use of anti-CTLA-4 antibodyand surrogate therapeutic endpoint can be used for induction oftherapeutic autoimmune responses to treat patients having aninappropriate accumulation of other self-antigens, such as amyloiddeposits, including Aβ in Alzheimer's disease, cytokines such as TNFα,and IgE.

EXAMPLES

The present invention is also described by means of the followingexamples. However, the use of these or other examples anywhere in thespecification is illustrative only and in no way limits the scope andmeaning of the invention or of any exemplified term. Likewise, theinvention is not limited to any particular preferred embodimentsdescribed herein. Indeed, many modifications and variations of theinvention may be apparent to those skilled in the art upon reading thisspecification and can be made without departing from its spirit andscope. The invention is therefore to be limited only by the terms of theappended claims along with the full scope of equivalents to which theclaims are entitled.

Example 1 Results from a Clinical Trial of Anti-CTLA-4 Antibody withMelanoma Peptides/IFA for Resected Stages III/IV Melanoma

Nineteen patients with resected Stage III (2 patients) or IV (17patients) melanoma received doses (0.3, 1 and 3 mg/kg) of CTLA-4antibody 10D1 with each injection of gp100/tyrosinase/MART-1 peptidevaccine with incomplete Freund's adjuvant (IFA). The patients wereHLA-A2⁺ and had no prior treatment with IFN-α. The tumors were IHCpositive for gp100, tyrosinase and/or MART-1. Exclusion criteriaincluded autoimmune disease and prior treatment with MDX-010, or MART-1,gp100 and tyrosinase peptides. The tyrosinase 368-376 (370D), MART-126-35 (27L) and gp100209-217 (210M) peptides each differed from wildtype by one amino acid modification to increase HLA binding.

Anti-CTLA-4 antibody and the peptide vaccines were administered every 4weeks for 6 weeks, then every 3 months×2 (8 doses total). The peptidevaccines were administered subcutaneously at 1 mg each per doseemulsified in IFA. Three cohorts received 0.3, 1.0, or 3.0 mg/kg IV ofanti-CTLA-4 antibody.

TABLE 1 Disease Status and Drug Related Serious Adverse Events PriorTreatment (I = immunotherapy, Dosing Sex/ C = chemotherapy, Diseasecohort Patient Age R = radiotherapy) SAE's status 0.3 mg/kg 1 M/43 I, CNone 2 F/40 I, C None Relapsed 3 F/69 I, C None 4 F/22 I, C None 5 M/57I, C None Relapsed 6 F/54 None None Relapsed 7 F/41 R, I None 1.0 mg/kg8 M/58 None None Relapsed 9 F/69 R None 10 M/64 None None Relapsed 11F/43 I None Relapsed 12 M/48 I None 13 F/59 None None 19 M/56 None Grade3 diarrhea, grade 3 fever, grade 3 bloody stool, bilateral uveitis 3.0mg/kg 14 M/36 None None 15 F/42 I, C Grade 3 16 M/40 I diarrhea 17 M/54I None 18 M/41 None Grade 3 diarrhea Grade 3 abdominal pain, grade 2diarrhea

TABLE 2 Dosing Summary Status Dose Number of doses Average doses Lost tomg/kg n 1 2 3 4 5 6 7 per patient follow-up Tox PD 0.3 7 1 4 2 5.8 4 31.0 7 1 1 1 1 1 2 4.3 1* 3 3.0 5 1 1 1 1 1 4.0 3* *MDX-010 discontinueddue to autoimmune manifestations, peptides continued

SAE's included:

-   1) A 40 year old male developed grade 2 diarrhea (two days) and    grade 3 abdominal pain (ten days) after the first infusion of 3    mg/kg of anti-CTLA-4 antibody. CT scan of the abdomen and pelvis    showed thickening and inflammation of the terminal ileum and cecal    wall. A repeat CT scan four days later showed resolution of the    abnormal findings. The patient was treated for his symptoms, which    resolved 16 days from onset.-   2) A 42 year old female developed grade 3 diarrhea six days after    the second infusion of 3 mg/kg of anti-CTLA-4 antibody. The patient    was treated with IV hydration and antibiotics.-   3) A 54 year old male developed grade 3 diarrhea after the third    infusion of 3 mg/kg of anti-CTLA-4 antibody. The patient's stool was    positive for white blood cells (WBC). The diarrhea was treated with    antibiotics and resolved 8 days after onset.-   4) A 56 year old male developed grade 3 bloody diarrhea and    bilateral uveitis after the second infusion of 1 mg/kg of    anti-CTLA-4 antibody. The patient's stool was positive for WBC,    sigmoidoscopy revealed inflamed bowel wall, and CT scan showed a    thickened cecal wall. The patient had an almost immediate response    to treatment with oral and topical steroids. All symptoms resolved    after three months.

Conclusions

There were no relapses in the highest dose cohort. Anti-CTLA-4 antibodydose-dependent, organ-specific auto-immune-like adverse events wereobserved. These adverse events were manageable and reversible.

Example 2 Results from a Clinical Trial of Single-dose Anti-CTLA4Antibody in Patients with Surgically Unresectable Stage III or IVMelanoma

A Phase I open-label, multicenter clinical trial was conducted toevaluate the safety and pharmacokinetics of MAb 10D1 in seventeenpatients with progressive, unresectable, malignant melanoma. Median agewas 59 years (range 29-79). Nine patients had received priorimmunotherapy, six had prior radiation and five had prior chemotherapy.All patients received a single dose of 3 mg/kg of 10D1 intravenouslyover 90 minutes and were then followed for toxicity, pharmacokinetics,circulating T cell activation and clinical outcome. All infusions werecompleted with only mild adverse events. Seven patients had mild,reversible rashes or pruritis. Plasma levels of antibody persisted fromone to four months. There was no significant increase in activatedperipheral T cells and no evidence of clinical autoimmunity beyond themild rash. Two patients experienced a partial response includingresolution of three soft tissue masses and over 50% reduction of a lungmass. Furthermore, the patient experiencing the over 50% reduction inlung mass was a patient who previously had been treated with a melanomavaccine, suggesting that the anti-CTLA-4 antibody treatment was capableof activating a pre-existing memory response to the tumor. The resultsof this study indicate that anti-CTLA-4 treatment was well toleratedwith clear evidence of immunologic and anti-tumor activity.

TABLE 2 Summary of patient characteristics and results Best ResponseSerious Age Prior Status and Metastatic Areas of Adverse Patient Sex(yrs) Treatment¹ Duration² Disease Sites Response Events 1 F 66 R, C SDLung, liver 2 F 69 None PD Liver 3 M 43 C, I SD Lymph node 4 M 56 R, C,I PD Lymph node, bone, peri-anal 5 M 43 R, C, I PD Spleen, adrenal, CNS,lung, retroperitoneum, skin 6 M 58 R, I SD Lung, lymph node, CNS 7 M 49I PD Skin, lung, liver, lymph node 8 F 70 H PD Lung, skin, liver 9 F 79None PD Unknown 10 M 74 R, C PD Lymph node, Grade III lung, pancreas,atrial kidney fibrillation 11 M 76 None PR, 7 months Lymph node, Lymphskin, lung, node, abdomen skin, lung, abdomen 12 F 49 I¹ PR, 9 monthsLung, skin, Lung, pleural effusion skin, pleural effusion 13 M 52 C, IPD Lung, liver 14 M 61 C, I PD Lung 15 M 29 R, C SD Lymph node, lung 16M 70 None PD Skin, liver, spleen 17 F 63 None PD Lung, lymph node ¹H =hormonal, R = radiotherapy, C = chemotherapy, I = immunotherapy ²PD =progressive disease, PR = partial response, SD = stable disease¹Immunotherapy included IL-2 and dendritic cell vaccine.

Example 3 Results from a Clinical Trial of Anti-CTLA-4 Antibody inCombination with gp100 Peptide Vaccines

A. Cohort 1

Fourteen patients with progressive Stage IV melanoma receivedanti-CTLA-4 antibody 10D1 in conjunction with vaccination with twoHLA-A*0201-restricted gp100 peptides. Patient characteristics aresummarized in Table 3.

TABLE 3 Patient characteristics and results Best Metastatic Response²Areas Age/ Disease Prior and of Patient Sex Sites Therapy¹ DurationResponse SAE's 1 52/M Lung C, I PR (8+ months, Lung Grade 3 pruritis,continuing) Grade 3 diarrhea 2 40/F Lymph node H, C, I SD Grade 3rash/desquamation 3 39/M Skin, lymph None SD node, lung 4 55/F Skin I SD5 67/M Bone, liver, C, I, R PD lung, lymph node, skin, intramuscular 658/M Lung, skin I PD 7 48/M CNS³, lung I PD 8 48/M Lung, liver, C, I PDadrenal, lymph node, skin 9 52/M Lymph node I SD Grade 3 diarrhea 1062/M Lung, lymph C, I SD node 11 54/M Lung, CNS, skin None CR (7+months, Lung, Grade 3 hypopituitary/ continuing)⁴ CNS, confusion skin 1243/M Intraperitoneal, I SD Grade 4 LFT intramuscular, elevation⁵ skin 1349/F Lung, adrenal C, I CR (7+ months, Lung, Grade 3 continuing) adrenalrash/desquamation 14 63/M Lung, pelvis, None SD lymph node ¹C =chemotherapy, H = hormonal, I = immunotherapy, R = radiotherapy ²PD =progressive disease, SD = stable disease, PR = partial response, CR =complete response. ³CNS = central nervous system ⁴The patient'scondition appeared to worsen prior to showing a positive response ⁵LFT =liver function test

All patients were HLA*0201⁺ with a Karnofsky performance status ≧60%.Six patients had visceral metastases. The patients had no evidence ofautoimmune or immunodeficiency disease. All patients had prior surgeryfor their primary lesion. Six patients had prior chemotherapy. Elevenpatients had prior immunotherapy including interferon-α (Patients 2,5-8, 10, 12 and 13), low-dose IL-2 (Patients 2, 5 and 13), high-doseintravenous IL-2 (Patients 4, 7 and 8), whole cell melanoma vaccines(Patients 1, 2 and 6), NY-ESO-1 peptide vaccine (Patients 4 and 5), andGM-CSF (Patient 9). The patients had no prior gp100 immunization and hadno systemic therapy in the three weeks prior to treatment.

A treatment cycle was administered every three weeks, which consisted ofanti-CTLA-4 antibody 10D1 at 3 mg/kg administered intravenously over 90minutes followed by 1 mg of gp100:209-217(210M) peptide (IMDQVPFSV)emulsified in incomplete Freund's adjuvant (IFA) injected subcutaneouslyin one extremity and 1 mg of gp100:280-288(288V) peptide (YLEPGPVTV)emulsified in IFA injected subcutaneously in a second extremity(synthetic peptides provided by the National Cancer Institute CancerTherapy Evaluation Program). Patients underwent apheresis prior totreatment and three weeks following every two treatment cycles.Peripheral blood mononuclear cells (PBMC) were isolated byFicoll-Hypaque separation and cryopreserved in heat-inactivated human ABserum with 10% dimethyl sulphoxide and stored at −180° C. until furtheruse.

Clinical response was evaluated using computed axial tomography (CT) ofthe chest, abdomen and pelvis; and magnetic resonance imaging (MRI) ofthe brain. These imaging studies were performed within 4 weeks ofstarting treatment and then after every two treatment cycles. Additionalradiological studies were used as needed to evaluate disease sites. Thesum of the longest diameters of the tumors in each patient (World HealthOrganization RECIST criteria) was calculated before and after treatment.A partial response was defined as a decrease of at least 30%, but lessthan 100%, in the sum of the longest diameters of all evaluablemetastases lasting at least one month, and no new or enlarging tumors. Acomplete response was defined as a decrease of 100% in the sum of thelongest diameters of all evaluable metastases lasting at least onemonth, and no new tumors.

Patients were evaluated for autoimmune responses. Patients received anophthalmologic examination prior to treatment and three months followinginitiation treatment. All patients had negative serum blood tests priorto initiation of the study for thyroglobulin Ab, rheumatoid factor andanti-nuclear antibody. Human anti-human (anti-idiotypic) Ab, erythrocytesedimentation rate, anti-nuclear Ab, thyroid stimulating hormone andfree T4 levels were measured every three weeks during the study.

Plasma concentrations of MDX-010 were determined using standard ELISAwith microtiter wells coated with CTLA-4-Ig (R&D Systems, Minneapolis,Minnesota). Dilutions of plasma samples were incubated on the plates.Bound anti-CTLA-4 Ab was detected with alkaline phosphatase-labeled goatanti-human IgG F(ab)-specific probe, which was developed with p-NPPsubstrate.

A twelve-day in vitro sensitization assay, which is more sensitive thanELISPOT or tetramer assays, was used to assess immunologic reactivity inall eleven patients with PBMC available for testing. (Rosenberg, S. A.et al., Immunologic and therapeutic evaluation of a synthetic peptidevaccine for the treatment of patients with metastatic melanoma. Nat.Med. 4:321-327 (1998)) Cryopreserved PBMC were thawed and cultured incomplete Iscove's-based media with 10% heat-inactivated human AB serumwith 1 μM of native gp100:209-217 or gp100:280-288 peptide and 300 IU/mlIL-2. Cells were harvested 11 to 13 days after initiation of the cultureand co-incubated with tumor cells or peptide-pulsed T2 cells overnight.Interferon-γ (IFN-γ) release in the supernatant was measured usingcommercial ELISA assays (Pierce-Endogen, Rockford, Ill.). All elevenpatients exhibited successful immunization against the nativegp100:209-217 peptide after one to four treatment cycles. Six patientswere successfully immunized against the native gp100:280-288 peptide.

Flow cytometry analyses were performed after Fc-receptor blocking andstaining with antibodies (BD Biosciences, San Diego, Calif.) ortetramers (Beckman Coulter Immunomics, San Diego, Calif.). Surfacemarker expression on PBMC of nine patients before and after two cyclesof treatment was compared. HLA-DR (an activation marker) expression wassignificantly increased on post-therapy CD3⁺CD4⁺ cells (P=0.0004; pairedt-test) and CD3⁺CD4⁺ (presumably CD8⁺) cells (P=0.04). CD3⁺CD4⁺ cellsalso showed significantly increased expression of CD45RO (a memory cellmarker) post-therapy (P=0.04). The percent of cell populationsexpressing CD69, CD25 and CTLA-4 did not change.

Patients 1, 11 and 13 were responders. (Table 15) Patient 1 hadshrinkage of a solitary lung lesion after two treatment cycles. Patient13 had complete resolution of a solitary lung lesion and an adrenallesion. Patient 11 had 31 lung lesions, two subcutaneous lesions and onebrain lesion. The brain lesion grew from 0.5 cm to approximately 1.0 cmafter two treatment cycles. Following three additional treatment cycles,Patient 11 had complete resolution of all lesions, including the brainlesion.

Grade 1/2 adverse events included diarrhea (Patients 3, 5 and 14), skinrash (Patient 14), pulmonary infiltrates and mild pleuritic chest pain(Patient 4) and vitiligo (Patients 2 and 6).

Six patients developed seven Grade 3 adverse events including dermatitis(Patients 1, 2 and 13), colitis/entercolitis (Patients 1 and 9),hypophysitis (inflammation of the pituitary gland) (Patient 11), andhepatitis (Patient 12). All patients recovered following discontinuationof treatment and the administration of supportive care and/or steroidtherapy. There were no relapses or subsequent autoimmune events.

Autoimmune screening blood tests were normal except for Patients 5 and12 who developed anti-nuclear antibody (ANA).

The mean peak of MDX-010 after the first dose was 72±33 μg/mL and thetrough before the second dose was 12±7 μg/mL. No clear correlationbetween plasma concentrations or antibody clearance and tumor regressionor toxicity was observed.

Patient 1, a partial responder, developed a generalized erythematousmaculopapular rash associated with severe pruritis one week after thesecond treatment cycle. A skin biopsy showed perivascular lymphocyticand eosinophilic infiltrate, papillary dermal edema and epidermalspongiosis consistent with an allergic drug eruption. Two days later,Patient 1 developed severe diarrhea and he was given IV hydration.Gastrointestinal endoscopy and biopsy showed multiple areas ofinflammation and mucosal ulceration with marked duodenal and coloniclymphocytosis, plasmacytosis and eosinophilia. Immunohistochemicalstudies indicated a predominance of CD3⁺ cells (CD8⁺>CD4⁺ cells) in theinflammatory infiltrate, polyclonality of the plasma cells and increasedMHC-1 and HLA-DR expression in the vasculature and epithelium.Autoimmune enterocolitis was diagnosed and the patient was treated withIV methylprednisolone. The patient had marked clinical improvementwithin 24 hours and the steroids were tapered over five days. Thepatient had no relapse of symptoms.

Patient 2 developed mild generalized pruritis one week after the firstcycle of treatment, which progressed over the following two weeks tosevere, circumferential, erythematous macular rash on the extremitieswhere she had received the vaccine injections (right arm and left leg).Skin biopsy showed epidermal spongiosis, significant papillary dermaledema, and a prominent lymphocytic and eosinophilic infiltrate withvascular involvement as seen in collagen autoimmunity. Patient 2 wastreated symptomatically with hydroxyzine and diphenhydramine. The rashcleared after several weeks. The patient developed vitiligo on bothupper extremities over the following three weeks.

Patient 9 developed diarrhea 11 days after the second cycle oftreatment. Endoscopy showed pan-colitis. Colonic biopsy showed severeinflammation with marked cellular infiltration and crypt abscesses.Immunohistochemical studies demonstrated that the majority ofinfiltrating lymphocytes were CD3⁺ (with a predominance of CD4⁺ cells),the plasma cells were polyclonal and epithelial MHC-1 and HLA-DRexpression were increased. The patient's diarrhea improved with IVmethylprednisolone treatment and was controlled with a slow taper oforal dexamethasone.

Patient 11, a complete responder, developed personality changes andmemory problems after receiving the fourth treatment cycle. MRI of thebrain showed disappearance of a left temporal metastasis and no otherabnormalities. Further evaluation showed undetectable levels of thyroidstimulating hormone, free T4, adrenocorticotropic hormone, growthhormone, prolactin and testosterone suggestive of pan-hypopituitarism. Arepeat, focused MRI showed the pituitary gland to be at the upper sizelimit of normal. High dose steroids were not used because the patienthad a complete clinical response. The patient received replacement dosesof thyroxine, testosterone and hydrocortisone. The patient's personalityand memory abnormalities resolved. A follow-up MRI six weeks latershowed a slight decrease in the size of the pituitary gland.

Patient 12 developed abnormal liver enzymes and anti-nuclear antibodieson routine blood tests done three weeks after the third cycle oftreatment. Liver biopsy showed acute hepatitis with numerous foci oflobular inflammation consisting mainly of lymphocytes.Immunohistochemical studies revealed a predominately CD3⁺ cellularinfiltrate with CD4⁺ cells mainly in the peri-portal areas and CD8⁺cells mainly in the hepatic lobules. Over the following two weeks, thepatient's alanine aminotransferase levels peaked at 2860 U/L (normal is6-41) and aspartate aminotransferase levels peaked at 1193 U/L (normalis 9-34). Low dose oral prednisone therapy was instituted and all valuesdecreased to normal over the following four months.

Patient 13, a partial responder, developed a severe generalizederythematous and pruritic rash one week after receiving the fourth cycleof treatment. Skin biopsy showed a perivascular lymphocytic infiltrationwith abundant eosinophils in the superficial dermis. Immunohistochemicalstudies revealed mainly CD3⁺ cells (CD4⁺>CD8⁺ cells). Lymphocytescultured from a biopsy of the rash were all CD8⁺ and 97% reacted withgp100:209-217:HLA-A*0201 tetramer. The rash slowly resolved withhydroxyzine treatment.

B. Cohort 2

The protocol of cohort 2 was the same as cohort 1 except that followingan initial loading dose of 3 mg/ kg of anti-CTLA-4 antibody, thepatients in cohort 2 received doses of 1 mg/kg of anti-CTLA-4 antibodyevery three weeks in combination with the peptide vaccines. The cohort 2study is on-going. To date, three of 24 patients (13%) have had anobjective tumor response and two of 24 patients have had SAE's (8%).

TABLE 4 Summary of patient characteristics and results Best ResponsePrior Status and Metastatic Areas of Patient Sex Age Treatment¹Duration² Disease Sites Response SAE's 15 F 54 C PR (2+ months) Lung,lymph Lung, Grade 3 node Lymph node diarrhea 16 M 39 R, I PD Lymph node,liver, lung, skin 17 M 48 None PD Adrenal, CNS, Grade 3 lung, lymphdiarrhea node, skin 18 M 32 R, C, I PD Liver, lung, lymph node 19 F 60None PR (2+ months) CNS, CNS, gallbladder, gallbladder, lung, lymphlung, lymph node, skin node, skin 20 M 62 I Not Adrenal, bone, availablelymph node, (N/A)³ spleen 21 M 50 None N/A Lung 22 M 50 None PD Bone,lymph node, lung 23 M 64 R, C, I N/A Intraperitoneal, liver, lung 24 M62 I PR (1+ month) Lung, intramuscular, skin 25 F 61 I N/A Lymph node 26F 61 None N/A Lymph node 27 M 21 C, I N/A Unknown 28 F 45 R, C, I N/AUnknown 29 F 63 R, I N/A Lymph node, skin 30 F 59 I N/A Unknown 31 M 56C, I N/A Unknown 32 M 57 I N/A Unknown ¹H = hormonal, R = radiotherapy,C = chemotherapy, I = immunotherapy ²PD = progressive disease, PR =partial response, SD = stable disease ³Patient response data is notavailable at this time.

Conclusion

This study demonstrated that clinical responses to anti-CTLA-4 antibodyin combination with melanoma peptide vaccines strongly correlates withthe occurrence of autoimmune-like adverse side-effects. Four of eight(50%) patients with autoimmune-like serious adverse effects had aclinical response. Only 2 of 28 patients (7%) had a response in theabsence of any serious autoimmune-like adverse effect.

Example 4 Results from a Clinical Trial of Anti-CTLA-4 Antibody inCombination with the Approved Regimen for MELACINE®

Thirteen patients with malignant melanoma were administered anti-CTLA-4antibody 10D1 (3 mg/kg×2 doses 8 weeks apart) in combination with theapproved regimen for MELACINE® (including cyclophosphamide). Noobjective responses and no serious adverse events were observed.

Example 5 Statistical Analysis of the Results of Example 3 (a ClinicalTrial of Anti-CTLA4 Antibody in Combination with gp100 Peptide Vaccines

Six of the 38 patients had an objective tumor response. Four of thesesix responders (66.7%) had autoimmune serious adverse events. Only fourof the 32 patients who did not respond had autoimmune serious adverseevents (12.5%).

TABLE 5 Distribution of Autoimmune Serious Adverse Events (n = 38)Patients with Patients without Autoimmune Autoimmune SAE SAENon-responders 4 28 Responders 4 2

A continuity-adjusted Chi-square test and Fisher's exact test wereperformed to examine the autoimmune incidence rate differed betweenresponders and non-responders. A statistically significant correlationwas found between patients that respond and patients that developautoimmune serious adverse events using both the Chi-square test(p=0.0146) and the Fisher's exact test (p=0.0116).

Example 6 Statistical Analysis of the Results of Examples 2, 3 and 4

Eight of the 68 patients had an objective tumor response. Four of theeight responders (50%) had autoimmune serious adverse events. Only fourof the 60 patients who did not respond (6.7%) had autoimmune seriousadverse events.

TABLE 6 Distribution of Autoimmune Serious Adverse Events Patients withPatients without Autoimmune Autoimmune SAE SAE Non-responders 4 56Responders 4 4

A continuity-adjusted Chi-square test and Fisher's exact test wereperformed to examine the autoimmune incidence rate difference betweenresponders and non-responders. A statistically significant correlationwas found between patients that respond and patients that developautoimmune serious adverse events using both the Chi-square test(p=0.0028) and the Fisher's exact test (p=0.0049).

REFERENCES CITED

Numerous references, including patents, patent applications and variouspublications, are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the inventiondescribed herein. All references cited and discussed in thisspecification are incorporated herein by reference in their entirety andto the same extent as if each reference was individually incorporated byreference.

1. A method for treating cancer in a patient in need of such treatment,which method comprises administering an anti-CTLA-4 antibody in a dosagesufficient to induce a breakthrough event and detecting the breakthroughevent.
 2. The method of claim 1 wherein the breakthrough event is anautoimmune response.
 3. The method of claim 1 wherein the breakthroughevent is a severe, life-threatening, or disabling adverse event.
 4. Themethod of claim 1, wherein the breakthrough event is induced byadministering escalating doses of anti-CTLA-4 antibody.
 5. The method ofclaim 1, wherein the breakthrough event is induced by administeringanti-CTLA-4 at decreasing dosage intervals.
 6. The method of claim 1,wherein the breakthrough event comprises a manifestation of non-tumorrelated autoimmunity.
 7. The method of claim 1 wherein the cancer is animmunologically sensitive tumor.
 8. The method of claim 1 wherein thecancer is malignant melanoma.
 9. A method for treating melanoma in apatient in need of such treatment, which method comprises administeringanti-CTLA-4 antibody 10D1 in a dosage sufficient to induce abreakthrough event and detecting the breakthrough event.
 10. The methodof claim 9 wherein the breakthrough event is an autoimmune response. 11.The method of claim 9 wherein the breakthrough event is a severe,life-threatening, or disabling adverse event.
 12. The method of claim11, wherein the breakthrough event is selected from the group consistingof diarrhea, enterocolitis, dermatitis, hypophysitis,panhypopituitarism, rash, and pruritis.
 13. The method of claim 9,wherein the breakthrough event is induced by administering escalatingdoses of anti-CTLA-4 antibody.
 14. The method of claim 9, wherein thebreakthrough event is induced by administering anti-CTLA-4 at decreasingdosage intervals.
 15. The method of claim 9, wherein the breakthroughevent is selected from the group consisting of dermatitis, vitiligo, andenterocolitis.
 16. The method of claim 1, where an additionaltherapeutic agent for treating the cancer is administered.
 17. Themethod of claim 1, where an additional therapeutic agent for treatingthe breakthrough event is administered.
 18. The method of claim 3,wherein the breakthrough event is selected from the group consisting ofdiarrhea, enterocolitis, dermatitis, hypophysitis, panhypopituitarism,rash, and pruritis.
 19. The method of claim 1, wherein the breakthroughevent is selected from the group consisting of dermatitis, vitiligo, andenterocolitis.